The broad, ling-term objectives of this research proposal are to develop general methods for protein structure analysis by NMR and to expand our understanding of the details of molecular recognition in protein: RNA complexes. This is a long-term project combining expertise in moleculr biology, molecular biophysics, and modern NMR spectroscopy. The work focuses on NMR studies of proteins and protein: RNA complexes in the 15 - 30 kD range. Technology for determining high resolution structures in this size range will be developed primarily o domains os a biomedically-important RNA- binding protein, the nonstructural protein 1 (NS1 protein) from influenza virus and complexes between the RNA-binding domain of the NS1 protein and its specific RNA target molecules. Influenza virus infection is a major human health problem in the United States and throughout the world. The NS1 protein plays a central role in the life cycle of the influenza virus and is potentially an important target for the rational design of antiviral drugs. We will characterize the three- eimensional structures and internal dynamics of the RNA-binding domains from two types (A and B) of human influenza virus. Specific RNA oligonucleotide molecules will be produced, and their binding epitopes on the surfaces of these NS1 proteins will be mapped by heteronuclear NMR. In addition, we will extend this work to dtudies of the 18kD effector domain of the influenza A virus NS1 protein, which will be expressed in E.coli and studies by NMR. This domain is responsible for regulating the translocation of mRNA from the nucleus into the cytosol, thereby regulating the expression of viral and cellular proteins during the infection cycle. This work will provide new insights into the mechansims by which proteins bind to RNAm and details of structure-function relationships in the NS1 protein from influenza virus. Neuropeptide Y (NPY), a 36, residue peptide amide initially isolated from porcine brain, is a member of a family of homologous hormones including peptike YY (PYY) and pancreatic polypeptide. It is the most abundant and widely distributed neuropeptide present in the mamalian central and peripheral nervous system. NPY has been implicated in a wide variety of central and peripheral functions. Of these, the most striking are its effects on feeding, blood pressure and cardiac contractility. Investigations to date suggest that NPY is the most potent orexigenic substance known. Its potent vasoconstrictor effects have also led to the classification of NPY among the most potent vasocpressor peptides isolated to date. While hypothalamic NPY levels and/or its mRNA are increased in obese rats, it is decreased in anorectic tumor bearing rats. Also, plasma NPY levels are elevated in patients with congestive heart failure (CHF) and pheochromocytoma, and in hypertensive rats. Moreover, NPY actions on feeding, blood pressor and contractility are mediated by Y-1-like, Y-1 and Y-3 subtypes, respecitively. These obsevations indicate that highly selective NPY analogs could be designed and synthesized to treat a variety of disorders. Our systematic investigations dircted towards these goals during the past none years have led to the identification of lower molecular weight strucyural moieties, T-54 and T-190 using classical SAR strategy. Our approach will include: 1) altteration of the hydrophobic/ hydrophilic / steric/ionic characteristics of each amino acid; 2) substitution with unnatural/D-amino acids, and 3) imposing structural constraints via cyclization, cyclic amino acids and backbone modifications. T-54 and T-190 analogs will be screened for their effects on feedings (Y-1 like) and on SK-N-MC cells (Y-1) , respectively. Similarly, we have already determined that the introduction of pseudobond in the C-terminal region of our cardiac NPY receptor antagonist, NPY (18-36), can impart high affinity and selectivity. Therefore, it is also proposed to continue to synthesize and investigate the receptor affinities and the effects on cardiac adenylate cyclase activity of NPY (18-36) analogs. These investigations are expected to result in the development of highly selective and long acting analogs for a variety of NPY receptors. These analogs will have great significance both in fundamental studies as well as in treating (or in developing drugs for) obesity, hypertension and failing heart.
